Technical Abstract:
Pestiviruses, members of the family Flaviviridae, encode one large polyprotein that is co- and post-translationally processed by viral and host-cell proteases. The nonstructural N-terminal protease (Npro) possesses autocatalytic activity to cleave itself from the remainder of the viral polyprotein. The function of the Npro has yet to be determined. Npro nucleotide sequence was obtained from Genbank or by sequence analysis for 5 isolates of each of the following Pestiviruses: classical swine fever virus, border disease virus, bovine viral diarrhea virus type 1, and bovine viral diarrhea virus type 2. The Npro sequences of all 20 isolates were compared and conserved amino acid residues were noted. Site-directed mutagenesis of conserved regions of the bovine viral diarrhea virus strain 890 Npro was accomplished by PCR using mutagenic primers, with mutated products being cloned into pBluescript for T7 RNA polymerase directed transcription. Autoprotease activity of mutant Npro proteins was evaluated based on the presence or absence of a 20 kDa cleaved protein (Npro) from the 35 kDa precursor after in vitro translation. Mutations at amino acids Gly-67, Cys-69, His-130, and Cys-168 resulted in loss of Npro cleavage. These results have indicated possible functional amino acid residues.